PURPOSE: When faced with a potential threat to health, the immune system has to answer two major questions. The first is "shall I respond?" Immunologists have long assumed that the immune system responded against all ?non-self? and tolerated all ?self? entities. 13 years ago, however, I proposed the Danger model, which assumes that the immune system's function is to discriminate between dangerous and harmless things rather than self and non-self. We have found that this model also leads to a new view of the second question ?What type of immune reaction should I produce?? Although most immunologists assume that the immune system tailors the type (class) of immune response to the pathogen it is fighting, we now think that it tailors the response to the tissue in which the response originates. Ultimately, we believe that the immune system is controlled by the tissues it is meant to protect, rather than by any particular cell or system belonging to the immune system itself.[unreadable] Because this model has tremendous implications for subjects like transplantation, cancer, neonatal and adult vaccines, parasitology, and autoimmunity, we have been testing its basic premises and its applicability in several areas. Some of our research is in its infancy, while other aspects are further along in development.[unreadable] [unreadable] THIS YEAR'S RESULTS[unreadable] [unreadable] 1) BIODEFENSE AND NEONATAL IMMUNITY Although there is a concerted national effort to find vaccines for potential bio-terrorism agents, little is being done to protect the nation?s infants. Most adult vaccines do not work for infants less than 6mo old, and many do not work for babies <1yr. Because we cannot leave our children behind, we have been analyzing the immune responses of newborns.[unreadable] A) THE 'INHIBITORY' EFFECTOF MATERNAL ANTIBODIES: In 1996 we showed that newborn mice were immunologically competent (contrary to common opinion), as long as they were properly immunized (Science 271:1723-1726). We have now been looking at a second myth, namely that the presence of maternal antibody inhibits newborn responses. We found that neonatal mice respond perfectly well to the model antigen Ovalbumin (OVA), even if they have received passive anti-OVA antibody from their mothers. The immunity in babies from immunized mothers lasts for at least 1yr, and does not fade more rapidly than that of babies from control mothers. Thus, in mice, maternal antibodies are not inhibitory.[unreadable] B) MATERNAL ANTIBODIES AND ANTHRAX: To see if our results with the model antigen, OVA, hold for real infections, we are studying the response to Anthrax. In preliminary studies to find the right vaccine dose, we found (astonishingly!) that the current test for adult immunity (neutralizing antibody) is misleading. Mice immunized with Anthrax protective antigen (PA) in adjuvant make high titers of antibody (as seen on ELISA tests) but no ?neutralizing? antibody. Yet they are completely protected from challenge with live Anthrax. This result may radically change how to vaccinate soldiers, and will also change how we analyze the responses of newborn mice.[unreadable] [unreadable] 2) TRANSPLANTATION TOLERANCE When neonatal hearts are grafted into immunodeficient (RAG KO) mice and allowed to heal for 9mo, the recipients become populated with small numbers of T cells from the grafted hearts and become resistant to rejection. We are now analyzing the microchimeric T cell populations to see if they have a role in the induction/maintenance of tolerance. [unreadable] [unreadable] 3) CD4 T CELLS CLEAR TUMORS In the process of studying how CD4 helper T cells collaborate with CD8 effectors to clear tumors, we found that the CD4 T cells are better at clearing tumors than CD8 cells. Against six out of six tumors, from five different tissues, CD4 effectors were more potent than CD8s. Searching for the mechanism, we found that the CD4 T cells partner with NK cells. We are now studying this partnership to determine which cell does what. (Perez-Diez, et al (2006?) CD4 Cells Can Be More Efficient at Tumor Rejection Than CD8 Cells submitted to Immunity)[unreadable] [unreadable] 4) DENDRITIC CELLS & IL-12p70 It is currently thought that dendritic cells control the effector class of an immune response by producing IL-12p70 upon stimulation by LPS. We found that this is incorrect (see bibliography publication #1). It is the p40 subunit, not p70 that is made. IL-12 p70 is only produced by dendritic cells that encounter previously activated T cells. It is also currently thought that dendritic cells become ?exhausted? within 24 hours of stimulation by LPS. This is also incorrect. The dendritic cells, while resistant to restimulation by LPS, can produce IL-12p70 when re-stimulated by activated T cells. Thus dendritic cells are not in control of immune effector class, but instead relay signals from the cells with which they are in contact.[unreadable] [unreadable] 6) IMMUNITY & HEALING The Danger model led us to the view that tissues influence the effector class of immunity in order to prevent immune-mediated local damage. To determine whether wounded tissues are affected by the immune system, we punched small holes in the ears of mice and measured the healing rate. We found that young mice heal slowly while older mice, surprisingly, quickly regenerate both the epithelium and cartilage so well that there is eventually no visible scar. B6 KO mice lacking B cells heal even better than WT mice. We are now asking if it is the presence of B cells or of antibody that discourages healing.[unreadable] [unreadable] 7) TOLERANCE TO LATE-APPEARING ANTIGENS[unreadable] One of the problems with the self-non-self model is that it does not account for tolerance to antigens that appear late in life, such as the milk proteins of lactating mothers (eg. alpha lactalbumin, beta lactoglobulin, casein etc.). To see if tolerance to these proteins is established in the thymus, or perhaps by fetal exposure during pregnancy or neonatal exposure during lactation, we used two different model systems in which we can obtain adult animals that carry a particular milk-protein gene but which have not previously encountered the protein, either across the placenta, or by drinking the milk as babies.[unreadable] A) HUMAN ALPHA LACTABLUMIN KNOCK-IN MICE: We obtained mixed-background mice that had their own alpha lactalbumin (mALAC) genes replaced with the human allele (hALAC), and bred them for 22 generations to B10.Br. To make experimental animals, we bred normal B10.Br females to B10.Br hALAC males. The F1 progeny carry both human and mouse alleles of ALAC but have only been exposed to mALAC from their mothers. When immunized to hALAC after weaning but before puberty, these mice (both males and females) made good T cell and antibody responses. Thus they were not rendered tolerant by thymic expression. When bred, the immunized females showed no autoimmune reactions to their mammary tissue. Neither did the levels of antibodies to hALAC drop or the subclasses of antibodies change. We are now in the process of making T cell clones from immunized female mice that do and do not express the hALAC gene, to make TCR Tg mice from these to follow the fate of immunized self-reactive T cells as the mice lactate through several pregnancies.[unreadable] B) SHEEP: Because fetal sheep have a 6 layer placenta, they receive no large maternal proteins. At birth, for example, they have no maternal antibody and need colostrum to obtain maternal protection. Lambs from sheep dairies (eg. Chattham Dairy, NY) are raised on cow?s milk replacer (as the sheep milk is used for cheese). We immunized such lambs after weaning and before puberty and found that, like the hALAC mice, they made good responses and did not manifest autoimmune disease when they were later bred and lactated. Biopsies from the udders showed no sign of infiltration or destruction.[unreadable] [unreadable] Thus the immune system has a mechanism to instigate tolerance to proteins that appear late in life, even after immunization.